Sputum cellularity in pulmonary tuberculosis: A comparative study between HIV-positive and -negative individuals pptx

017 – 023, February, 2010 Available online http://www.academicjournals.org/JAHR ©2010 Academic Journals Full Length Research Paper Sputum cellularity in pulmonary tuberculosis: A compar

Journal of AIDS and HIV Research Vol 2(2) pp 017 – 023, February, 2010

Available online http://www.academicjournals.org/JAHR

©2010 Academic Journals

Full Length Research Paper

Sputum cellularity in pulmonary tuberculosis: A

comparative study between HIV-positive and -negative

individuals Rosemeri Maurici da Silva1*, Paula Stocco1, Maria Luiza Bazzo2 and Mariana Chagas2

Accepted 15 December, 2009

To compare sputum cellularity between HIV-positive and -negative individuals with pulmonary tuberculosis A cross-sectional study was conducted in patients with pulmonary tuberculosis Sputum samples were collected and processed within two hours after collection The absolute number of

percentage of eosinophils, lymphocytes, macrophages and neutrophils and total cellularity and viability were determined Comparisons of the means of each cell type were held in a significance level of 95% (p < 0.05) Pearson’s correlation coefficient between the identified cell types was calculated Results: Assessment was performed in a cohort of 40 subjects, mean age 40 years, 77.5% male, 70% Caucasian, 40% HIV-positive (mean age 35.9 years) Mean percentage viability in the samples was 56.1% The average value of squamous cells was 58.8 Mean percentages of cells were: 33.7% neutrophils, 1.7%

patterns was present in 72.5% of cases Pearson’s correlation coefficient was 0.08 (p < 0.01) between absolute counts of eosinophils and lymphocytes, eosinophils and macrophages and macrophages and neutrophils Inverse relationship was observed between the percentage of macrophages and neutrophils There was no statistically significant difference between cell count of HIVpositive and -negative individuals

Key words: Sputum, tuberculosis, HIV

INTRODUCTION

Tuberculosis is a chronic infectious disease caused by

Mycobacterium tuberculosis bacillus (Koch bacillus),

whose main characteristic is the preference for lung

parenchyma and transmission from person to person,

which occurs by inhalation of microorganism infected

particles (Brasil Ministério da Saúde Coordenação

Nacional de DST/AIDS, 1999)

With the advent of the acquired immunodeficiency

syndrome (AIDS), recognized in 1981, a profound impact

*Corresponding author Email: rosemaurici@hotmail.com

on the global problem of tuberculosis occurred, which changed its epidemiology and particularly, its control became more difficult Tuberculosis kills approximately two million people annually and measures to control this disease are vulnerable to early diagnosis, resistance to drugs used for its treatment as well as the socioeconomic conditions of populations at risk (Brasil Ministério da Saúde Coordenação Nacional de DST/AIDS, 1999; Duncan et al., 1996)

M tuberculosis is a facultative intracellular bacterium; its replication process and the way it is carried through the host during the course of infection are not completely defined It is believed that macrophages are the main

018 J AIDS HIV Res

tuberculosis is found in extracellular environment in the

stages of lung cavitation It is not clear how the bacterium

adapts specifically to the lungs at the expense of other

tissues and how the bacterium survives and grows in

phagocytes of macrophages and other cells (Brasil

Ministério da Saúde Fundação Nacional de Saúde,

2002)

Human immunodeficiency virus (HIV) infection is a

leading risk factor for the development of disease in

individuals previously infected by the bacillus While the

chance of an infection progressing to TB disease in

immunocompetent individuals is 10% over their life span,

in HIV-infected individuals that is likely to be 8 to 10%

every year Moreover, it is one of the first and major

complications among HIV-infected individuals, appearing

before other common infections (Brasil Ministério da

Saúde Fundação Nacional de Saúde, 2002; Davis et al.,

1993)

In 1999, 10.7 million people co-infected with TB/HIV

were identified, which represents 0.18% of the world

population In Brazil, of the 40.7 million infected with

tuberculosis, about 300 thousand were co-infected with

HIV (Brasil Ministério da Saúde Coordenação Nacional

is an important differential diagnosis when lung

secretions are sent for laboratory examination Currently,

there is little information on sputum cytology of patients

with pulmonary tuberculosis (Brasil Ministério da Saúde

Fundação Nacional de Saúde, 2002)

Radiological alterations of patients with TB/HIV

manifestations of TB are atypical At the beginning of HIV

to that in immunocompetent patients, with typical

reactivation pattern and with areas of alveolar

consolidation at the apex, posterior segments of the

upper lobes and superior segments of the lower lobes,

often associated with cavitation (Boiselle et al., 2002;

Haramati and Jenny-Avital, 1998; Shah et al., 1997;

Keiper et al., 1995; Post et al., 1995; Naidich and

McGuinness, 1991; Pitchenik and Rubinson, 1985;

Pizzichini et al., 1996) In patients who are in advanced

documented, compared to immunocompetent patients,

such as mediastinal and/or hilar lymph nodes and in

some cases, no radiographic alterations (Haramati and

Jenny-Avital, 1998; Shah et al., 1997; Keiper et al., 1995;

Naidich and McGuinness, 1991; Pitchenik and Rubinson,

1985) Tuberculosis infection in immunocompetent

individuals begins primarily as a non-specific

inflammatory reaction, progressing to a typical

count, granuloma formation does not occur (Botasso et al., 2007)

Although primarily cellular immunity is involved, other defects are also identified and play important role in the morbidity of HIV infection T lymphocytes are critical for the activation of B lymphocytes and subsequent production of immunoglobulins, which is compromised by the primary disorder of cellular immunity These systemic alterations are concomitant to the local alterations Subsystems of different T lymphocytes are involved in

Interferon-gamma production by cells appears to be fundamental for disease control Th1 cytokine response type is predominant in patients with mild and moderate forms of pulmonary tuberculosis, while Th2 type cytokine production prevails in more severe disease Studies show that patients with cavitary tuberculosis revealed the presence of IL-4 produced by Th2 system In contrast, Th1-type cytokines are found in cases of non-cavitary disease (Botasso et al., 2007)

The macrophages present CD4 antigens and can be directly infected by HIV In these cases the process of chemotaxis is also disturbed, resulting in decrease or even absence of granulomatous reaction In addition, there is a decrease in chemotaxis of polymorphonuclear cells (Davis et al., 1993) Therefore, alterations in sputum cellularity occur concurrently to alterations in peripheral

respiratory diseases in this specific group of patients, both in the radiological manifestations and in the tissue reactions where there is no granuloma formation due to decreased immunity (Davis et al., 1993)

During a recent infection with HIV, when the function of the immune system is relatively intact, sputum examina-tion of smear-positive for tuberculosis predominates In contrast, patients with advanced HIV infection with significant immunosuppression often present negative sputum examination results and disease disseminates Although the correlation between the significance of sputum examination and decrease in the immune system

HIV-positive patients with pulmonary tuberculosis is well do-cumented, the relationship between sputum examination and local immune response in the lungs is not clear (Mwandumba et al., 2008)

The study carried out by Belda and collaborators found the following results for sputum cellularity in healthy patients: cell viability was 89.7%, the proportion of eosinophils was 1.1%, neutrophils 64%, macrophages 86.1%, lymphocytes 2.6%, metachromatic cells 0.04% and epithelial cells 4.4% Female gender and atopy are associated with a significant elevation of eosinophils, male-to-female ratio was 0.3% and between atopic and non-atopic patients was 0.4% (Belda et al., 2000)

There is little information on sputum cytology in pulmonary tuberculosis A study carried out by Tani and

collaborators showed that alveolar macrophages are

always present, while neutrophils are present in 97.9% of

samples, usually in large numbers Lymphocytes were

found in 84.9% and eosinophils in 8.9%, usually in small

numbers Epithelial cells were found in 56.1% of

sam-ples, usually appearing in groups with oval and elongated

nucleus, along with a large vacuolated cytoplasm

Multinucleate giant cells were present in 40% of samples,

usually in small numbers and often associated with

epithelial cells Respiratory epithelium cells showed

changes in 20% of samples, which include grouped

columnar cells with hyperchromatic nucleus Squamous

metaplasia was observed in 19% of samples (Tani et al.,

1987)

This study was carried out to compare sputum

cellularity between HIV-positive and HIV-negative

individuals with pulmonary tuberculosis

MATERIALS AND METHODS

A cross-sectional study was performed at Hospital Nereu Ramos, in

Florianopolis, Santa Catarina, in which all patients over 14 years

with pulmonary tuberculosis admitted between November 2008 and

February 2009 were analyzed Patients who had pulmonary

comorbidity were excluded from the study, as well as those who

refused to sign the Term of Free and Informed Consent, or those

who were unable to produce sputum spontaneously HIV-infected

patients were considered those who had positive serology for HIV,

patients with pulmonary tuberculosis and those with identification of

M tuberculosis in respiratory samples (bronchoalveolar lavage

and/or lung and pleural biopsies)

Chest x-rays were classified according to alteration patterns in

alveolar consolidation, interstitial, pleural effusion, mass, nodule,

cavitation, mediastinal and/or hilar lymph nodes and their

associations Siemens X-ray device was used for postero-anterior

and lateral chest radiography, using 120 Kv and 3 to 6 mAs

Sputum samples were collected at morning and before breakfast

The sputum samples were processed within two hours after

collection An aliquot of sputum was treated with approximately 4

volumes of DTT (dithiothreitol) plus 4 volumes of PBS (phosphate

buffer) and filtered after homogenization Twenty microlitres of

filtrate were mixed with 20 µ l of 4% trypan blue This mixture was

placed in a Neubauer chamber where the absolute number of cells

(x 10 6 cells/ml) and the number of live and dead cells in a clear field

microscope with a 400x magnification were counted The

percentage of live and dead cells was resulting from the equation:

(number of live cells/total number of cells) × 100 The presence of

alveolar macrophages was determined by differential leukocyte

counting in the filtrate of the sputum sample The filtrates were

concentrated by citospin technique and the slides were stained by

the May-Grünwald/Giemsa method and viewed in clear field

microscope with a 1000x magnification The number of squamous

epithelial cells in a total of 400 cells and the number of eosinophils,

lymphocytes, macrophages and neutrophils on differential count of

100 cells was determined The absolute number and percentage of

each cell type was calculated based on the total number of cells

(Pizzichini et al., 1996; Botasso et al., 2007)

Each participant was registered in a form of inclusion and agreed

to participate by signing a Term of Free and Informed Consent

Database development and statistical analysis were performed

using SPSS version 16.0 ® software Data were summarized as

per-centage or mean, as indicated, and comparisons of means of each

cell type were performed by Student’s t-test, with a significance

Maurici da Silva et al 19

level at 95% (p < 0.05) The Pearson’s correlation coefficient for the identified cell types was also calculated

The research project was submitted to the Ethics Committee and Human Research at Unisul and approved under code number 09.005.4.01.III.

RESULTS

Forty consecutive individuals, 31 (77.5%) male, were evaluated Regarding ethnicity, 28 (70%) were Caucasian Mean age was 40 years (SD±12), ranging from 22 to 69 years

Of the participants, 16 (40%) were HIV positive and 24 (60%) were negative Mean age of HIV-positive individuals was 35.9 years (SD±7.16) and mean age of HIV-negative individuals was 42.8 years (SD±13.8) There was no statistically significant difference between mean age of HIV-positive and HIV-negative individuals (p 0.75)

Mean percentage of viability in sputum samples was 56.1% (SD±31%) The average value of squamous cells

in a total of 400 cells assessed was 58.8 (SD±157.7) Mean value of cells (× 106 cells/ml±SD) and mean percentage (±SD) in sputum samples were: neutrophils 0.9 ± 1.4 (33.7 ± 3.2%), eosinophils 0.03 ± 0.08 (1.7 ± 2.8%), macrophages 0.8 ± 1.3 (50.7 ± 20.3%), lymphocytes 0.2 ± 0.3 (12.3 ± 11.8%) and total cells 1.9 ± 2.5

Mean value and percentage of cells in sputum samples

of HIV-positive and HIV-negative individuals are shown in Table 1

There was a statistically significant difference in absolute counts of squamous cells when compared between HIV-positive and -negative individuals (p 0.028) There was no statistically significant difference between the other cell counts when compared between HIV-positive and -negative individuals (p > 0.05)

With regard to radiological alterations, the association between patterns was present in 29 (72.5%) of cases (10 HIV-positive and 19 HIV-negative), alveolar injury in 32 (80%) of cases (13 HIV-positive and 19-negative), interstitial lesion in 15 (37.5%) of cases (8 HIV-positive and 7 -negative), cavitation in 10 (25%) of cases (4 HIV-positive and 6 -negative), pleural effusion in 9 (22, 5%) of cases (3 HIV-positive and 6 -negative), atelectasis in 4 (10%) of cases (1 HIV-positive and 3 -negative), nodules

in 3 (7.5%) of cases (HIV-negative) and pneumothorax, adenomegaly and mass in 1 (2.5%) of cases, respectively (HIV-negative)

The average values and percentage of cells in sputum samples in accordance with the radiological alterations are shown in Table 2

Pearson’s correlation coefficient between the cell values in sputum samples is shown in Table 3

# x

6 /ml

The

022 J AIDS HIV Res

2007; Mwandumba et al., 2008)

considered the cut-off point between individuals who will

have the typical or atypical form of pulmonary

tuberculosis, because this value determines the acquired

immunodeficiency degree from the disease, that is,

indicating that patients had a very compromised

immunity

For average values of cells found in sputum samples

from individuals with and without HIV, there was a

statistically significant difference in the absolute count of

squamous cells, with higher values in HIV-positive

indivi-duals (p < 0.05) There is a probability that this figure

results from a higher contamination at collection of the

sputum sample of these patients (Efthimiadis et al., 1997)

There was no statistically significant difference between

the other cell counts when compared between

positive and negative individuals (p > 0.05)

peripheral blood, but this fact does not seem to occur in

the immune response that occurs in the lung tissue

Patho-physiology features of tuberculosis in this specific

group of patients may then be a consequence of

qualitative alterations of lymphocyte immune response

and not quantitative alterations as suggested in

peripheral blood In this study, lymphocytes found in

sputum samples were not typified, so there is no way to

know to which lymphocyte subpopulation they belong

The type of lymphocyte involved in immune response

determines its quality; further studies with this

metho-dology are needed to confirm or refute this hypothesis

(Botasso et al., 2007; Mwandumba et al., 2008; Deveci et

al., 2006; Nicod, 2007)

Radiological alterations in patients were distributed in

various patterns Pattern association was found in 72.5%

of cases Curvo-Semedo and collaborators reported the

coexistence of 30% between consolidation, cavitation

and lymph node (Curvo-Semedo et al., 2005) Cavitation

was present in 25% of cases Curve-Semedo’s study

reported that cavitation occurs in approximately 50% of

patients Lower results found in this study can be due to

the number of HIV-positive patients with low lymphocyte

CD4+ T-cell count in peri-pheral blood, which do not form

either cavitation or granules (Boiselle et al., 2002;

Haramati and Jenny-Avital, 1998; Shah et al., 1997;

Keiper et al., 1995)

Adenomegaly was reported in 2.5% of cases, which

corroborates the findings by Curvo-Semedo et al (2005),

who describe that mediastinal or hilar lymphnodes are

rarely found in post- primary di-sease, occurring in

approximately 5% of cases

The percentages of eosinophils were different in

individuals with interstitial lesion, being higher when the

lesion occurred (p < 0.05) This may be due to the quality

of immune response in patients with this type of injury when compared to other radiographic alterations (Boiselle et al., 2002; Haramati and Jenny-Avital, 1998; Shah et al., 1997; Keiper et al., 1995) Studies with larger sample sizes and methodology addressed to this topic in particular must be performed to confirm or refute this hypothesis Other differences between the cellular values found and the type of radio-logical alterations, although statistically significant, may not be highlighted due to the small number of patients with these alterations (adenomegaly, pneumothorax, nodules and atelectasis) Pearson’s correlation coefficient was 0.08 (p < 0.01) between eosinophil and lymphocyte abso-lute counts, indicating the concomitant increase in the two cell types

in the inflammatory response of pulmonary tuberculosis The same trend was observed between eosinophils and macrophages and between ma-crophages and neutrophils Inverse relationship was observed between the percentage of macrophages and neutrophils

Further studies with larger samples should be performed to confirm or refute the numerical trends presented here

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